人组织因子的密码子优化及其在毕赤酵母中的高水平发酵制备

优化人组织因子编码序列利用毕赤酵母表达系统高水平发酵制备重组人组织因子。将人组织因子胞外活性区(hTF219)编码序列进行密码子偏爱性、GC含量及富AT区的优化,优化序列电转入毕赤酵母表达系统;利用博莱霉素(Zeocin)梯度抗性平板筛选出高表达转化子;在1L发酵罐中采用混合碳源连续补料方式发酵制备重组蛋白。替换hTF219编码cDNA的165个碱基,获得的优化序列与原始序列的相似性为75%;在Zeocin浓度为500μg/mL的YPD平板上筛选得到高表达转化子tfPP-502,该重组菌株以组成型方式分泌表达重组人组织因子,在1L发酵罐中发酵110 h胞外总蛋白含量为6.27 g/L,目的蛋白占比约80%。在毕赤酵母表达系统中实现了重组人组织因子的高水平发酵制备获得的重组菌株可用于重组人组织因子的大规模发酵制备。

Codon Optimization and High Level Fermentation of Human Tissue Factor in Pichia Pastoris

To optimize the coding sequence of human tissue factor and to produce the recombinant human tissue factor(rhTF) by high level fermentation with Pichia pastoris expression system,the coding sequence of human tissue factor extracellular active region(hTF219) was optimized for expression with codon preference,GC content and AT rich region.Then the optimized sequence was transferred into Pichia pastoris expression system using electroporation.High expression transformants were screened out on YPD plates with increasing concentration of Zeocin,and was fermented in a 1-L fermentor to produce the recombinant hTF using continuous feeding style with mixed carbon sources.The optimized cDNA sequence of hTF219 had 165 bases replaced.The consistency between the optimized sequence and the original sequence was 75%.One high expression transformant tfPP-502 was screened on YPD plate with Zeocin concentration of 500 μg/mL.The recombinant strain could secreted express recombinant human tissue factor in a constitutive manner.And the total extracellular protein content was 6.27 g/L in 1-L fermentor for 110 hours,in which the target protein accounted for about 80%.High level fermentation of recombinant human tissue factor was obtained in Pichia pastoris expression system.The recombinant strain could be used for large-scale fermentation of human tissue factor.